Hormones are biological messengers. They are synthesized by specific tissues (glands) and are secreted into the blood. The blood carries them to target cells where they act to alter the activities of the target cells.
Hormones are chemically diverse, and are generally categorized into three main groups: (1) small molecules derived from amino acids, for example thyroxine, (2) polypeptides or proteins, for example insulin and thyroid-stimulating hormone, and (3) molecules derived from cholesterol, for example steroids.
An important class of hormone is the thyroid hormones. Examples of thyroid hormones are thyroxine (T4) and triiodothyronine (T3). Both T4 and T3 enter cells and bind to intracellular receptors where they increase the metabolic capabilities of the cell by increasing mitochondria and mitochondrial enzymes.
Steroids make up another important class of hormones. Examples of steroid hormones include estrogens, progesterone and testosterone. Estrogen is the name of a group of hormones of which there are three principle forms, estrone, estradiol and estriol. Estrogens and progesterone cause the development of the female secondary sexual characteristics and develop and maintain the reproductive function. Testosterone develops and maintains the male secondary sex characteristics, promotes growth and formation of sperm. Steroids enter target cells and bind to intracellular receptors and then cause the production of mRNA coding for proteins that manifest the changes induced by steroids.
The accurate analysis and quantification of hormones is becoming more important. For example, estrogen and estrogen like compounds are playing an ever-increasing role in today's society through hormone replacement therapy. Also, the analysis and quantification of estrogen and estrogenike compounds helps in the management of estrogen-related diseases, like breast cancer.
Presently, the common methods of hormone analysis use immunoassay techniques. Table 1 lists the common hormones and the current methods for their analysis.
For example, estriol is analyzed by a radioimmunoassay utilizing radiolabelled antigen (iodine 125) in competition with unlabelled estriol in the sample, for a known amount of antibody. The assay is read using a gamma counter.
Androstenedione is analyzed using an enzyme immunoassay comprising horseradish peroxidase. Unlabeled antigen in the sample is in competition with enzyme labeled antigen for a fixed number of antibody binding sites. The assay is read using a microtitre plate enzyme immunoassay reader.
Several hormones are currently analyzed using a chemiluminescent immunoassay. For example, progesterone, testosterone, cortisol and T3 are analyzed using this method. The assay utilizes an assay-specific antibody-coated bead. The assay is read using a photon counter.
However, the current immunoassays are disadvantageous for the following reasons:                (1) Immunoassays are specific to one hormone, therefore every hormone must be analyzed separately.        (2) Numerous kits must be purchased and procedures must be learned for each hormone being analyzed.        (3) Various instruments to read the results from the immunoassays must be purchased. For example, the analysis of estriol and progesterone from a sample requires both a gamma counter and a photon counter.        (4) The kits for the assays can be expensive.        (5) The current immunoassays lack specificity and may show approximately 15 fold difference in results using kits from different manufacturers [1]. Table 2 provides the mean low and high values for a number of steroids using different immunoassays currently available, illustrating their lack of specificity.        (6) The procedures involve many steps and can take a significant amount of time.        (7) In the case of a radioimmunoassay, precautions are necessary because of the radioisotopes involved.        
More recently, hormones have been analysed and quantified by mass spectrometry. However, there are several disadvantages to these methods.
For example, a method of analyzing urinary testosterone and dihydrotestosterone glucuronides using electrospray tandem mass spectrometry has been described [2]. The method involves a complex system employing high performance liquid chromatography (HPLC) and a three-column two-switching valve. The shortcomings include the following: (i) the hormone glucuronides were analyzed, not the hormones, (ii) the method is applicable to urine only and (iii) only two analytes were analysed simultaneously, (iv) the limit of detection (LOD) was 200 pg ml−1 for testosterone and the limit of quantification was 10 ug L−1 for dihydrotestosterone and (v) the method is complex.
Another publication discloses a method for the determination of estradiol in bovine plasma by an ion trap gas chromatography-tandem mass spectrometry technique [3]. The shortcomings include the following: (i) only one analyte was analyzed, (ii) 4 ml of plasma was required for the analysis of one analyte, (iii) the limit of detection was 5 pg ml−1, and (iv) derivation was required because the method employs gas chromatography. Unfortunately, the analysis of estrogens by mass spectrometry is problematic, because they show low sensitivity under conventional electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) techniques. Steroid compounds generally lack chemical groups with high proton affinity, so the protonation reaction that normally leads to the formation of the analytical ion is difficult to produce with the standard ESI and APCI sources.
A method for analysis of 17-hydroxyprogesterone by HPLC electrospray ionization tandem mass spectrometry from dried blood spots has also been described [4]. However, this method analyses only one analyte at a time, and requires liquid-liquid extraction, which is laborious and time consuming, with sample extraction alone taking 50 minutes to complete.
Finally, a gas chromatography mass spectrometry method to analyze the production rates of testosterone and dihydrosterone has been disclosed [5].
TABLE 1Methods and instruments for steroid and thyroid hormones [1]PercentageANALYTEof UseInstrumentMETHODAndrostenedione35%DSL solidEIA11-Deoxycortisol50%ICN Immuchem DARIADHEA Sulfate39%DPC ImmuliteECIAEstradiol16%Bayer ADVIA CentaurFIAEstriol, unconjugated25%DSL liquidRIAEstriol, Total50%DPC Coat-a-CountRIA17-Hydroxyprogesterone51%DPC Coat-a-CountRIAProgesterone23%Bayer ADVIA CentaurCIATestosterone29%Bayer ADVIA CentaurCIATestosterone, Free65%DPC Coat-a-CountRIAAldosterone76%DPC Coat-a-CountRIACortisol25%Bayer ADVIA CentaurCIAT329%Abbott AxsymFPIAT3, Free31%Bayer ADVIA CentaurCIAT430%Abbott AxsymFPIAT4, Free34%Abbott AxsymFPIARIA: RadioimmunoassayEIA: Enzyme Linked ImmunoassayFPIA: Fluorescence Polarization Immunoassay